Chapter title |
Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca 2+ -ATPase by Competitive Inhibition of [γ- 32 P]TNP-8N 3 -ATP Photolabeling
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Chapter number | 22 |
Book title |
P-Type ATPases
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Published in |
Methods in molecular biology, January 2016
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DOI | 10.1007/978-1-4939-3179-8_22 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3178-1, 978-1-4939-3179-8
|
Authors |
Johannes D. Clausen, David B. McIntosh, David G. Woolley, Jens Peter Andersen |
Abstract |
The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements. |
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